This kind of processes comprises bringing a porous or non-porous adsorbent matrix that exposes on its outer and/or inner (pore) surfaces a structure that is capable of binding to a desired substance into contact with an aqueous liquid sample derived from the appropriate biological material. The conditions are selected such that the desired substance becomes bound to the structure. In a subsequent step the substance is desorbed/eluted from the matrix and collected. Between the adsorption/binding step and the desorption/elution step there may be one or more washing steps that primarily will remove loosely held material derived from the sample. This latter material may for instance be non-adsorbed/non-bound material present in the liquid within the pores and/or in the interstitial liquid if the matrix is a bed of particles.
In order to make this kind of processes economically feasible there is mostly an imperative need to reuse the adsorbent matrix in several cycles of the same process, e.g. more than 50 cycles, such as more than 75 or 100 cycles. This means that the adsorbent matrix shall not be altered to any significant degree during the individual steps and/or cycles. Factors such as dynamic binding capacity, break through or total availability capacities for the desired substance, rigidity, porosity etc should be essentially constant between the cycles.
In a typical sample to be processed there are also compounds other than the desired substance that will attach to the adsorbent matrix during the adsorption step. One way of getting rid of these other material prior to a subsequent cycle has been to use solutions of guanidinium hydrochloride, urea, aqueous sodium hydroxide or hydrochloride at high concentrations.
Typical hydrolysis sensitive matrices are based on silica and/or saccharide structure. Typical binding structures that are sensitive to hydrolysis may contain peptide, saccharide and/or nucleic acid structure. Most critical are matrices having proteineous ligands. For binding structures/matrices that are sufficiently inert towards hydrolysis, the method of choice has been cleaning with aqueous sodium hydroxide (0.1 M or more such as more than 0.5 M or more than 1.0 M) when disturbing amounts of contaminants have assembled on the adsorbent matrix, typically after several cycles. In this latter case each cycle has in addition comprised a milder cleaning or regeneration step, for instance with a concentrated solution of a compound that compete with the substance for binding to the structure (concentrated salt solution when the binding is based on ion exchange). Alternatively regeneration/cleaning has taken place with acid solutions (e.g. 0.1 M or such as more than 0.5 M HCl). For hydrolysis sensitive materials the agents of choice have been concentrated solutions of urea or guanidinium hydrochloride.
In particular liquid samples containing adherent substances such as lipids, lipoproteins, hydrophobic proteins and other lipoid substances are problematic because they have a severe tendency to stick to the matrices thereby clogging inter-particle as well as intra-particle pores and/or otherwise blocking the binding structures. It has previously been suggested to clean adsorbent matrices from this kind of material by treatment with pure solvents such as ethanol and isopropanol. The use of organic solvents tends to create a new problem because they often precipitate salts and proteins. Even if organic solvents assist in getting rid of adherent material they will increase the risk for clogging by precipitates.
Concentrated solutions of guanidinium hydrochloride are highly corrosive meaning that they may create severe problems on reactors, tubings and connections, in particular if made of steel. In addition there are costs associated with wastes containing these agents and/or with their reuse. Concentrated acid or alkaline solutions, such as 0.1 to 0.5 M hydrochloride or 0.5 to 1 M sodium hydroxide are highly hydrolytic, and therefore can only be used to clean structure/matrices highly inert to hydrolysis.
The prior use of concentrated ethanol and isopropanol, which means in concentrations above 50% (v/v), also presents an explosion problem.
Furthermore, use of the concentrated regeneration solutions known in the art, such as 3 to 6 molar urea or guanidinium hydrochloride is caused in a further problem. The use of concentrated regeneration solutions due to high buffer consumption, especially when the chromatographic process is performed in a fluidized (=expanded) bed mode (EB mode). Displacement of the high concentrated (=high density) regeneration solution by a low density buffer, which is needed for re-use of the chromatography matrix, results in turbulences within the column. The flow direction in fluidized EB mode columns is from bottom to top. Therefore, the more denser solution tends to mix with the more lower density buffer due to gravity. As a result, displacement of the more denser solution is triggered by dilution and not by plug flow behavior, which leads to a significant higher buffer consumption.